Induction of apoptosis in human pancreatic MiaPaCa-2 cells through the loss of mitochondrial membrane potential (ΔΨm) by Gentiana kurroo root extract and LC-ESI-MS analysis of its principal constituents

Phytomedicine 20 (2013) 723–733

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Induction of apoptosis in human pancreatic MiaPaCa-2 cells through the loss of mitochondrial membrane potential ( m ) by Gentiana kurroo root extract and LC-ESI-MS analysis of its principal constituents Bilal A. Wani a , D. Ramamoorthy a , Manzoor A. Rather b,∗ , N. Arumugam c , Asif Khurshid Qazi d , Rabiya Majeed d , Abid Hamid d,∗ , Showkat A. Ganie e , Bashir A. Ganai e,∗ , Rajneesh Anand f , Ajai P. Gupta f a

Department of Ecology and Environmental Sciences, Pondicherry University, Puducherry 605 014, India Natural Product Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Srinagar 190005, India c Department of Biotechnology, Pondicherry University, Puducherry 605 014, India d Cancer Pharmacology Division, Indian Institute of Integrative Medicine, Canal Road, Jammu, India e Department of Biochemistry, University of Kashmir, Srinagar 190 006, Kashmir, India f Quality Control and Quality Assurance Division, Indian Institute of Integrative Medicine, Canal Road, Jammu, India b

a r t i c l e

i n f o

Keywords: Gentiana kurroo Royle LC–ESI-MSMS Gentiopicroside Iridoid glycosides Cell-cycle arrest Anti-proliferative activity Apoptosis

a b s t r a c t The objective of the current study was to evaluate the methanolic root extract of Gentiana kurroo for antioxidant and antiproliferative activities as well as to study the effect of the extract on the induction of apoptosis in human pancreatic cancer cell line (MiaPaCa-2). The extract exerted significant antioxidant activity as verified by DPPH, hydroxyl radical, lipid peroxidation and protective oxidative DNA damage assays. The results were comparable to standard antioxidants like ␣-tocopherol, catechin and BHT used in such experiments. Antioxidant potential of G. kurroo may be attributed to the presence of high phenolic and flavonoid content (73 ± 1.02 and 46 ± 2.05 mg/g extract respectively). The anti-proliferative property of Gentiana kurroo root extract was determined by sulphorhodamine B (SRB) assay against Human colon cancer cell line (HCT-116), Lung carcinoma cell line (A-549), Pancreatic cancer cell line (MiaPaCa-2), Lung cancer cell line (HOP-62) and acute monocytic leukaemia cell line (THP-1). G. kurroo root extract inhibited cancer cell growth depending upon the cell line used and in a dose dependent manner. The extract induced potent apoptotic effects in MiaPaCa-2 cells. The population of apoptotic cells increased from 11.4% in case of control to 49.6% at 100 ␮g/ml of G. kurroo root extract. The extract also induced a remarkable decrease in mitochondrial membrane potential ( m) leading to apoptosis of cancer cells used. The main chemical constituents identified by the liquid chromatography–tandem mass spectrometry (LC–ESI-MSMS) were found to be iridoid glucosides (iridoids and secoiridoids), xanthones and flavonoids. Iridoid glucosides are the bitter principles of Gentiana species. Loganic acid, Sweroside, Swertiamarin, Gentiopicroside, Gentisin, Isogentisin, Gentioside, Norswertianolin, Swertianolin, 4 -O␤-d-glucosyl-6 -O-(4-O-␤-d-glucosylcaffeoyl)-linearoside and Swertisin were the principal compounds present in the methanol root extract of G. kurroo. © 2013 Elsevier GmbH. All rights reserved.

Introduction Cancer is the second major cause of death worldwide, with increasing death rate every year despite of the extensive research focused on new treatment and prevention. Cancer develops via multistep carcinogenesis process involving various cellular physiological systems like cell signalling and apoptosis making it a very complex disease (Reichert and Wenger 2008). Most of the currently

∗ Corresponding authors. E-mail addresses: [email protected], [email protected] (M.A. Rather), [email protected] (A. Hamid), [email protected] (B.A. Ganai). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.01.011

used anticancer drugs which have been obtained either by synthesis of new compounds or from natural sources or by structural modification of natural compounds, are toxic to normal cells in addition to cancer cells and therefore, have substantial side effects. Hence, there is a continuous search for new chemotherapeutic drugs which may act as “magic bullets” targeting only cancer cells. A successful anti-cancer agent is one that kills cancer cells without causing too much damage to normal cells. This ideal situation is achievable by the induction of apoptosis in malignant cells. Thus, apoptosis modulation may be a key factor in the prevention or cancer treatment. Recently, induction of apoptosis in cancer cells has been a new target for innovative mechanism based drug discovery (Carnesecchi et al. 2001). Thus, it is very important to isolate

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and screen apoptotic inducers from the natural sources especially plants which synthesize a vast diversity of compounds involving nature’s combinatorial chemistry. Chemotherapeutic agents from plants consist of a vast array of compounds with diverse mechanism of action, but their ultimate tendency of inducing apoptosis in cancer cells may represent a unifying idea for the chemopreventive mechanism. The World Health Organization (WHO) has estimated that more than 75% of the population of Asian and African countries still rely on traditional medicine for prevention and treatment of diseases because of the high cost of western pharmaceuticals. Traditional medicine mostly involves the use of plant extracts. In the last few decades, natural products especially plant derived molecules have become an increasingly important source of potential anticancer agents which frequently seem to be more effective and/or less toxic. In view of the spectacular biodiversity of the planet (microbes, plants, animals, and marine organisms), a promising future for natural products in anti-cancer drug discovery seems likely; indeed, far more likely than synthetic compounds. During the last 50 years, more than 100,000 compounds have been screened for anticancer effect but out of them only seven plantderived anticancer agents have been approved by Food and Drug Administration (FDA) (Xianghui and Zhiwen 2009). Epidemiological studies suggest that natural antioxidants such as ascorbic acid (vitamin C), tocopherols (vitamin E), ␤-carotene (provitamin A), anthocyanins and other polyphenol supplements are associated with a lower incidence of cancers and cancerrelated mortality (Fleischauer et al. 2003). Excess generation of ROS takes place under certain pathophysiological conditions and by exogenous agents like drugs, pollution, UV light and other xenobiotics. The endogenous defence mechanism present in the living organisms at times becomes incapable to scavenge them completely. The excessive ROS produced can cause damages to cellular biomolecules like lipids, proteins, enzymes, DNA and RNA, resulting in membrane fluidity (Dean and David 1993), that ultimately results in the development of degenerative diseases (Shahidi et al. 1992), including cardiovascular diseases, cancers, neurodegenerative, and inflammatory diseases. DNA damage is a common event in all living cells that is caused by free radicals. Gentiana kurroo Royle (Gentianaceae) is an endemic plant of north-western Himalayas. It is a perennial herb with a strong well developed rhizome bearing decumbent flowering stems, each with 1–4 blue flowers, growing along sub-alpine Himalayas at an altitude of 1500–3000 m (The Wealth of India 2000). In Kashmir, the plant is commonly known as Nilkanth. The roots of the plant are source of iridoid glycosides like gentiopicrine, gentiamarin and the alkaloid gentianin (Sharma et al. 1993). Plants belonging to the genus Gentiana are known for their bitter taste which is due to the presence of secoiridoids (e.g. swertiamarin, gentiopicroside, sweroside and amarogentin) (Jiang et al. 2005). The roots of the plant are used as bitter tonic, antiperiodic, expectorant, astringent, stomachic, anthelmintic, antipsychotic, anti-inflammatory, antibacterial, sedative and analgesic (Kirtikar and Basu 1935; Bilal et al. 2011a). Six known iridoid glucosides (6-cinnamoyl, 6-o-cinnamoyl, 6-o-vanilloyl, 6-o-feruloylcatalpol, aucubin and catalpol) have been isolated from its roots (Sarg et al. 1991). In our previous study on G. kurroo, we have reported GC–MS analysis of volatile aroma components from aerial parts of G. kurroo (Bilal et al. 2011b). As part of our ongoing research programme towards the screening of various medicinal and aromatic plants of Kashmir Himalayas for their anticancer potential (Rather et al. 2012; Dar et al. 2011, 2012; Fozia et al. 2011), we report here for the first time the antioxidant and cytotoxic activity of the methanolic root extract of Gentiana kurroo along with the identification of main chemical compounds by liquid chromatography–tandem mass spectrometry

(LC–ESI-MSMS). The results of the present study indicate that G. kurroo blocks cell cycle progression in a dose dependent manner. There was a significant increase in sub G0 /G1 cell population (hypodiploid DNA content), which may be possibly due to DNA fragmentation, resulting in apoptotic cell death. Materials and methods Chemicals Folin–Ciocalteu reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH), gallic acid, catechin, linolenic acid, RPMI-1640 medium, rhodamine-123, propidium iodide, streptomycin, foetal bovine serum (FBS), sodium bicarbonate, phosphate buffer saline(PBS), sulphorhodamine-B (SRB), trypsin, Paclitaxel, 5-fluorouracil, gentamycin sulphate were purchased from Sigma–Aldrich. Butylated hydroxytoluene (BHT), trichloroacetic acid (TCA), thiobarbituric acid (TBA), ferric chloride, hydrogen peroxide (H2 O2 ), Potassium ferricyanide, dimethylsulfoxide (DMSO) were obtained from Merck. All the reagents used were of analytical grade. Plant sampling Gentiana kurroo Royle was collected at flowering stage in July–August 2011 from lower reaches of Pir-panchal range of Kashmir Himalaya (latitude 34◦ 12 10 N; longitude 74◦ 51 36 E, at an altitude of 2150 m). The plants were collected randomly over an area of 500 m2 . The identity of plant was confirmed at the Centre of Plant Taxonomy and Biodiversity, University of Kashmir (Jammu and Kashmir, India). A specimen under voucher number KASHKU/BAW-780 was deposited in Kashmir University Herbaria for future reference. Preparation of extract Roots of the plant were cut, properly cleaned and dried under shade at room temperature, chopped and ground to fine powder using a mechanical blender and passed through 24 mesh sieve. Dried root powder (100 g) was packed in Soxhlet apparatus and extracted with methanol at 60–65 ◦ C for 2–3 hours. The extract was filtered through Whatman filter paper No. 1. The residue was discarded and the filtrate was collected. The filtrate was concentrated under reduced pressure at 40 ◦ C using Buchi rotavapor (R-215). The extract was dried, labelled and stored at 4 ◦ C in storage vials for experimental use. Determination of total phenolics Total phenolic content of methanolic root extract of Gentiana kurroo was determined by Folin–Ciocalteu method with some modifications. 200 ␮l of sample (1 mg/ml) was added to 100 ␮l diluted (1:10) Folin–Ciocalteu reagent and equilibrated for a few minutes. Then 800 ␮l of 2.5% aqueous Na2 CO3 was added and mixture was allowed to stand for 60 min at room temperature with intermittent shaking. Absorbance of the blue colour solution so obtained was measured at 765 nm in a spectrophotometer [Shimadzu UVPC1650 (Japan)]. Gallic acid was used as standard. The absorbance of solution was compared with gallic acid calibration curve. The total phenolic content was expressed as gallic acid equivalents (mg GAE/g dry weight), and the values presented as mean ± SD of triplicate analysis. Determination of total flavonoids Flavonoid content was determined by aluminium chloride colorimetric method. The principle of this method is based on

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the formation of flavonoid–aluminium complex that has max at 430 nm. For this 0.5 ml of plant extract (1 mg/ml) was mixed with 1.5 ml of methanol, 0.1 ml of 10% AlCl3 , 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. After incubation at room temperature for 15 min, the absorbance of the reaction mixture was measured at 430 nm. Total flavonoid content was expressed as catechin equivalents (mg CE/g dry weight) using a calibration curve of catechin as standard. DPPH free radical scavenging assay The free radical scavenging property of G. kurroo root extract was measured on the basis of proton or electron donating ability of the extract. The donated protons or electrons reduces the purple DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical to yellow coloured complex. 100 ␮l of different concentrations (100–700 ␮g/ml) was added to 1 ml of 0.5 mM DPPH solution in methanol. The mixture after shaking vigorously, was allowed to stand at room temperature for 30 min in dark. Absorbance of the mixture was read at 517 nm against methanol. The decrease in absorbance indicates increase in DPPH free radical scavenging potential. The percentage inhibition was calculated using the equation. DPPH free radical inhibition (%) =

Ac − As × 100 Ac

where, Ac is the absorbance of control and As is the absorbance of sample. ␣-Tocopherol was used as standard antioxidant and served as positive control. Hydroxyl radical scavenging assay Hydroxyl radical, generated from the Fe3+ –ascorbate–H2 O2 (Fenton reaction), was evaluated by degradation of deoxyribose that produced thiobarbituric acid reactive species (TBARS). The reaction mixture containing 25 mM deoxyribose, 10 mM ferric chloride, 100 mM ascorbic acid, 2.8 mM H2 O2 in 10 mM KH2 PO4 (pH 7.4) and various concentrations (10–100 ␮g/ml) of G. kurroo extract. The reaction mixture was incubated at 37 ◦ C for 1 h. Then 1 ml of 1% (w/v) thiobarbituric acid and 1 ml of 3% (w/v) trichloroacetic acid were added and incubated at 100 ◦ C for 20 min. The TBARS was measured spectrophotometrically at 532 nm. The results were expressed as percentage inhibition of deoxyribose oxidation, as determined by the following formula. Percent inhibition =

A−B × 100 A

where A was the malonaldehyde produced by Fenton reaction treated alone, and B was the malonaldehyde produced by addition of G. kurroo extract and ␣-tocopherol as standard antioxidant. Lipid peroxidation assay Lipid peroxidation of tissue homogenates was measured by using the thiobarbituric acid test, employing the method of Buege and Aust (1978). Tissue homogenate (0.5 ml) was mixed with 5 ␮l of 10 mM ethylene diamine tetraacetic acid (EDTA) and 1 ml thiobarbituric acid–trichloroacetic acid–chloridic acid (TBA–TCA–HCl) solution and placed in a boiling water bath for 15 min. After cooling, the flocculent precipitate was removed by centrifugation at 6000 rpm for 10 min. Absorbance of red TBA malonaldehyde complex was then measured at 532 nm. Malonaldehyde formed from the degradation of poly unsaturated fatty acids, was then calculated by using an extinction coefficient of 1.56 × 105 M−1 cm−1 .

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Protective effect on oxidative damage to DNA The Protective effect on oxidative damage to calf thymus DNA by G. kurroo root extract was performed following the method of Ghanta et al. (2007). Hydroxyl radicals generated by Fenton reaction were used to induce oxidative damage to DNA. The reaction mixture (15 ␮l) contained 25 mg of calf thymus DNA in 20.0 mM phosphate buffer saline (pH 7.4) and different concentrations of plant extract (10, 30, 50, 80 and 100 ␮g) were added and incubated with DNA for 15 min at room temperature. The oxidation was induced by treating DNA with 20 mM ferric nitrate and 100 mM ascorbic acid and incubated them for 1 h at 37 ◦ C. The reaction was terminated by the addition of the loading buffer bromophenol blue (0.25%) and glycerol (30%) and the mixture was subjected to gel electrophoresis in 0.7% agarose/TAE buffer run at 100 V. DNA was visualized and photographed by gel doc.

Cell lines and culture conditions Human colon cancer cell line (HCT-116), Lung carcinoma cell line (A-549), Pancreatic cancer cell line (MiaPaCa-2), Lung cancer cell line (HOP-62) and acute monocytic leukaemia cell line (THP-1) were procured from National Cancer Institute (NCI), Frederick, U.S.A and European Collection of cell culture (ECACC), UK. Cells were grown as monolayers in RPMI-1640/MEM medium supplemented with 10% heat inactivated foetal calf serum, 100 U/ml penicillin, 100 ␮g/ml streptomycin and sterilized by filtering through 0.2 ␮m filter in laminar air flow hood. Cells were grown in CO2 incubator (New Brunswick, Galaxy 170R, eppendroff) at 37 ◦ C in 5% CO2 with 98% relative humidity. In all experiments, cells were allowed to adhere and grow for 24 h in culture medium prior to exposure to crude extract.

Anti-proliferative assay The anti-proliferative property of Gentiana kurroo root extract was determined by sulpharhodamine B (SRB) assay as described by Monks et al. (1991). An aliquot of 100 ␮l of cell suspension (1 × 105 cells/well) was seeded in flat-bottomed 96-well plates and incubated overnight in a humidified atmosphere containing 5% CO2 set at 37 ◦ C. 100 ␮l crude extract at different concentrations (10–100 ␮g/ml). 5-Fluorouracil (20 ␮M) and Paclitaxel (1 ␮M) were added to cells as standard anticancer drugs. After 48 h of incubation, the cell growth was stopped by adding 50 ␮l of 50% trichloroacetic acid in each well and plates were further incubated at 4 ◦ C for an hour. The plates were washed with distilled water and air-dried. The cells were stained with 50 ␮l of SRB reagent and incubated at room temperature for 30 min. The plates were washed with 1% acetic acid to remove unbound SRB and allowed to dry overnight. 100 ␮l of 10 mM Tris–base were added to each well to solubilize the dye and stirrer for 5 min at room temperature. The optical density was recorded on ELISA reader at 570 nm. All the experiments were performed in triplicates and results were ±SD of three experiments. The percentage cell growth inhibition was calculated by following equation: Percent cell viability =

At × 100 Ac

Percent cell growth inhibition = (100 − percent cell viability) where At is the absorbance of treated cells and Ac is the Absorbance of control cells.

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DNA content and cell cycle phase distribution Human pancreatic cell line (MiaPaCa-2) was seeded in 6-well culture plate (2 × 105 /ml/well). Cells were incubated for 24 h and then treated with different concentrations (0, 30, 50 and 100 ␮g/ml) of plant extract. After 48 h of treatment, cells were harvested by centrifugation for 5 min at 1000 rpm, washed twice with PBS and fixed with 70% ethanol overnight at −20 ◦ C, and then stained with DNA staining solution (20 mg/ml PI, 0.1 mM EDTA, 10 ␮g/ml RNAase and 1% triton X-100 in PBS) for 30 min in dark. DNA content was measured by using a flow cytometer analysis system (BD FACS Aria). Data from 10,000 cells were collected for each data file. All the histograms were analysed using FACS Diva software. Mitochondrial membrane potential ( m ) for cellular energy status

Concentration (mg/g dry weight)

726

80 70 60 50 40 30 20 10

Liquid chromatography–tandem mass spectrometry (LC–ESI-MSMS)

fl av an o id s

T o ta l

T o ta l

p h en o lic s

0 Mitochondrial membrane potential ( m ) was measured using flow cytometry with 2 ␮M rhodamine-123 (Rh-123), cellpermeable cationic dye that preferentially enters mitochondria based on the highly negative mitochondrial membrane potential. Depolarization of the membrane results in the loss of Rhodamine-123 from the mitochondria and a decrease in intracellular fluorescence. MiaPaCa-2 cells (2 × 105 cells/ml/well) were seeded in 6-well culture plate and incubated for 24 h. Cells were treated with different concentration (0, 30, 50, 100 ␮g/ml) of plant extract for 48 h treatment. Rh-123 (10 ␮g/ml) was added 1 h before the termination of experiment, incubated at 37 ◦ C for 30 min and thereafter washed with PBS. The pellet collected by centrifugation, was resuspended in 300 ␮l of PBS. The florescence intensity of Rh123 in cells was analysed using flow cytometer set at 485 nm.

Fig. 1. Total phenolic and flavonoid content of Gentiana kurroo root extract. Each value represents the mean ± SD (n = 3). Total phenol content was expressed as mg gallic acid/g dried extract. Total flavonoid content was expressed as mg catechin/g dried extract.

GAE/g dry extract (Fig. 1). The flavonoid content was found to be 46 ± 2.05 mg equivalent of catechin/g dry extract. Flavonoids are known to exhibit a wide range of biological activities including cytotoxic, antioxidant, anti-inflammatory, analgesic, and antimicrobial activities. DPPH assay

LC–MS equipment (LC–MS QqQ-6410B Agilent Technologies) comprised a chromatographic system (1260 Infinity Agilent Technologies) coupled with an Agilent Triple Quad mass spectrometer fitted with an ESI source. MS conditions were the following: MS range 100–1200 Da, MSn spectra were obtained using both positive and negative modes, nebulizer gas 45 Psi, gas temperature 325 ◦ C, capillary voltage 4000 V. HPLC analysis was carried out by an Agilent 1260 infinity series. A Chromolith RP-18e column (4.6 mm ID, 50 mm length) (Merck) was used. Mobile phase consisted of (A) aqueous formic acid (0.1%) and (B) methanol. Gradient condition was; 0–8 min, linear gradient from 12% to 25% of B; 8–12 min, isocratic conditions at 25% of B; 12–16 min, linear gradient from 25% to 40% of B; 16–40 min, linear gradient from 40 to 50% of B, 40–50 min, linear gradient from 50 to 100% of B. Flow rate: 1 ml/min.

DPPH assay is a simple, rapid, reliable in vitro assay for determing free radical scavenging activity of any new drug. DPPH (1,1-diphenyl-2-picrylhydrazyl) is a stable purple free radical compound having a nitrogen as a free radical which can be easily quenched by a radical scavenger. In the presence of a proton radical scavenger or hydrogen donating antioxidants, DPPH radicals are transformed into a neutral yellow coloured complex DPPHH (1,1-diphenyl-2-picrylhydrazine) (Blois 1958). The degree of decolouration indicates the scavenging potential of antioxidant extract (Van et al. 1997). The crude extract was found to exhibit concentration dependent free radical scavenging activity (Fig. 2). The extract at 100 ␮g/ml produced 25.60% of DPPH scavenging effect, which increased up to 69.90% at 700 ␮g/ml. The results were comparable with the standard antioxidant ␣-tocopherol at the same concentration.

Results and discussion Hydroxyl radical scavenging assay Total phenolic and flavonoid content Polyphenols have received considerable attention because of their physiological function, including antioxidant, antimutagenic and antitumour activities (Othman et al. 2007). It is reported that phenolic compounds in plants possess strong antioxidant activity and help in protecting cells against oxidative damage caused by free radicals. The antioxidant phenolic compounds act as reducing agents, hydrogen donors and singlet oxygen quenchers and metal chelators. Folin–Ciocalteu method is one of the widely used method for finding the polyphenol contents (Prior et al. 2005). This assay in the present study showed that the phenolic content in the methanolic extract of G. kurroo was found to be 73 ± 1.02 mg

Hydroxyl radical (HO• ) is a highly reactive oxygen centred free radical formed in biological systems and is highly damaging species in free radical pathology, capable of damaging almost every molecule found in living cells (Hochestein and Atallah 1988). Hydroxyl radical attacks nucleotides in DNA and cause strand breakage, which contributes to carcinogenesis and mutagenesis. It initiates the lipid peroxidation process by abstracting hydrogen atoms from polyunsaturated fatty acids of membrane lipids (Aruoma 1999). Gentiana kurroo rhizome extract exhibited concentration dependent scavenging activity against hydroxyl radical generated by Fenton reaction system (Fig. 3). The extract showed 53.93 ± 1.53% hydroxyl radical scavenging activity at 100 ␮g/ml

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100

Gentiana Kurroo Vitamin E

80 75

Gentiana Kurroo

80

70

BHT

70

65

60

60

% Inhibition

% Inhibition

90

727

50 40 30 20

55 50 45 40 35

10

30

0 100

200

300

400

500

600

700

Concentration (µg/ml)

25 20

Fig. 2. Effect of G. kurroo root extract and known antioxidant on DPPH radical scavenging activity. The results represent mean ± S.D of 3 separate experiments. Absorbance at 517 nm.

15 50

100

150

200

250

Concentration (µg/ml) concentration. Hydroxyl radical scavenging ability of extract is directly related to the prevention of propagation of the process of lipid peroxidation and is a good scavenger of reactive oxygen species. Microsomal lipid peroxidation Lipid peroxidation is an auto-catalytic free radical mediated destructive process, whereby polyunsaturated fatty acids in biomembranes undergo degradation to form lipid hydroperoxides, which latter decompose to form a wide variety of products including ketones, fatty acids, low molecular weight hydrocarbons, alkenols and alkanals, in particular malonaldehyde (MDA) (Zeyuan et al. 1998). The reduction of MDA production would indicate lipid peroxidation inhibition. Fe2+ /Ascorbate/H2 O2 model system were used to initiate lipid peroxidation in rat liver microsomes. MDA forms a pink chromogen with TBA which gives maximum absorbance at 535 nm. Initiation of lipid peroxidation by ferric nitrate, ascorbic acid and H2 O2 takes place either through ferryl–perferryl complex or through hydroxyl radical (HO• ) in the Fenton reaction. Gentiana kurroo rhizome extract inhibited MDA

100

Gentiana Kurroo Vitamin E

90

% Inhibition

80 70

Fig. 4. Effect of methanol extract of G. kurroo and known antioxidant BHT on microsomal lipid peroxidation.

formation, and thus lipid peroxidation in liver microsomes in concentration dependent manner (Fig. 4). The extract produced 54.30% LPO inhibition at 250 ␮g/ml concentration. BHT at the same concentration produced 74% LPO inhibition. The beneficial effect of G. kurroo on lipid peroxidation may be attributed to its high polyphenolic content. Protective effect on oxidative damage to DNA G. kurroo root extract showed potential antioxidant activity as depicted by its ability to inhibit DNA damage induced by hydroxyl radical generated by FeSO4 and H2 O2 in Fenton reaction. DNA is susceptible to oxidative damage and hydroxyl radicals oxidize guanosine or thymine to 8-hydroxyl-2- deoxyguanosine and thymine glycol which change DNA and lead to mutagenesis and carcinogenesis (Ames et al. 1993). The protective effect of G. kurroo methanol extract on calf thymus DNA is shown in the Fig. 5. Hydroxyl radicals generated in presence of ferric nitrate/ascorbic acid/H2 O2 were found to induce DNA strand breaks and helps the DNA molecule to run fast (Lane-2). G. kurroo extract at 10–100 ␮g/ml concentration offered complete protection to DNA damage induced by hydroxyl radicals in calf thymus DNA in concentration dependent manner (Lane 3–7). Catechin (10 ␮g/ml) was used as standard (Lane-8).

60

Anti-proliferative assay

50

Sulpharhodamine B (SRB) assay is based on the measurement of cellular protein content. The assay relies on the uptake of the negatively charged pink aminoxanthene dye (SRB) by basic amino acid proteins under mild acidic conditions (Vanicha and Kanyawim 2006). Treatment of cells with G. kurroo extract (10–100 ␮g/ml) produced dose-dependent anti-proliferation effect on different human cancer cell lines HCT-116, THP-1, A-549, MiaPaCa-2 and HOP-62 (Table 1). It was observed that the extract at higher concentration (100 ␮g/ml) was the most potent with a high percentage cell growth inhibition of 87 ± 2.40 and 74 ± 2.57 against human acute monocytic leukaemia (THP-1) and human colon (HCT-116) cancer cell lines respectively. At lower concentration (10 ␮g/ml), percent cell growth inhibition observed was 39 ± 4.55 and 37 ± 1.80

40 30 20 10 0

20

40

60

80

100

Concentration (µg/ml) Fig. 3. Effect of G. kurroo root extract and Vitamin E on hydroxyl radical scavenging activity. Values represents the mean ± SD (n = 3).

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Fig. 5. Protective effect of G. kurroo root extract on oxidative damage to calf thymus DNA. Lane 1: native calf thymus DNA. Lane 2: DNA + 20 mM ferric nitrate + 100 mM ascorbic acid + 30 mM H2 O2. Lane 3: DNA + 20 mM ferric nitrate + 100 mM ascorbic acid + 30 mM H2 O2 + 10 ␮g of extract. Lane 4: DNA + 20 mM ferric nitrate + 100 mM ascorbic acid + 30 mM H2 O2 + 30 ␮g of extract. Lane 5: DNA + 20 mM ferric nitrate + 100 mM ascorbic acid + 30 mM H2 O2 + 50 ␮g of extract. Lane 6: DNA + 20 mM ferric nitrate + 100 mM ascorbic acid + 30 mM H2 O2 + 80 ␮g of extract. Lane 7: DNA + 20 mM ferric nitrate + 100 mM ascorbic acid + 30 mM H2 O2 + 100 ␮g of extract. Lane 8: DNA + 20 mM ferric nitrate + 100 mM ascorbic acid + 30 mM H2 O2 + 10 ␮g of Catechin. Table 1 Anticancer activity of methanolic root extract of Gentiana kurroo. Cell line type Tissue type Gentiana kurroo

5-FU Paclitaxel

HCT-116 Colon 100 ␮g/ml 50 ␮g/ml 10 ␮g/ml 20 ␮M 1 ␮M

74 ± 53 ± 22 ± 67 ± –

2.57 1.46 3.53 1.87

A-549 Lung 65 ± 48 ± 34 ± – 70 ±

2.39 4.09 3.85 2.45

THP-1 Leukaemia 87 ± 61 ± 37 ± 67 ± –

2.40 3.35 1.80 1.56

HOP-62 Lung

MIA-Pa-Ca Pancreatic

62 ± 31 ± 21 ± – 72 ±

59 ± 3.25 45 ± 3.86 39 ± 4.55 – –

1.74 2.96 3.15 2.78

The results represent mean ± S.D of three experiments.

Fig. 6. Effect of G. kurroo root extract on cell cycle phase distribution of human pancreatic cancer cell line (MiaPaCa-2). Flow cytometric analysis of MiaPaCa-2 cells after propidium iodide staining. Cells were incubated for 48 h in presence of G. kurroo methanolic extract at (0, 30, 50 and 100 ␮g/ml) concentration. Figures show the representative staining profile of one of two similar experiments. P1 is the population of apoptotic cells, which increases from 11.4% in case of control to 49.6% in case of 100 ␮g/ml of G. kurroo methanolic root extract.

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Fig. 7. Effect of Gentiana kurroo root extract on mitochondrial membrane potential loss ( m ). Gentiana kurroo root extract induced loss of mitochondrial membrane potential ( m ) in human pancreatic cancer cell line (MiaPaCa-2) incubated with extract at different concentrations (0, 30 50 and 100 ␮g/ml) in 6 well plate for 48 h treatment.

in human pancreatic cancer cell line (MiaPaCa-2) and human acute monocytic leukaemia (THP-1) cell lines respectively. The findings of our study shows that methanolic root extract of G. kurroo inhibits the proliferation of different human cancer cell lines in concentration dependent and cell line specific manner. Involvement of different polyphenolic compounds that act synergistically to display anti-proliferative activity has been earlier suggested by Yang et al. (2009). DNA content and cell cycle phase distribution Cell-cycle arrest in cancer cells has become a major indicator of anticancer event. The effect of G. kurroo root extract on cell cycle progression was analysed by flow cytometry. Propidium iodide (PI) staining of MiaPaCa-2 cell line exposed to different concentrations (0, 30, 50 and 100 ␮g/ml) of plant extract for 48 h induces apoptotic induction. Apoptotic cells were selected as shrunken

cells with degraded chromatin, high side-scatter (SSC) and low forward-scatter (FSC) properties (Bachir et al. 2012). Fig. 6 shows that the proportion of sub G0 /G1 cell population increases from 11.4% in case of control to 49.6% in case of 100 ␮g/ml of G. kurroo extract. The results indicate that G. kurroo blocks cell cycle progression in concentration dependent manner. There was a significant increase in sub G0 /G1 cell population (hypodiploid DNA content), which may be possibly due to DNA fragmentation, resulting in apoptotic cell death. The inhibition of cell cycle progression may be one of the molecular events associated with the selective anti-cancer efficacy of G. kurroo as is depicted by MiaPaCa-2 cell line. Loss of mitochondrial membrane potential ( m ) Disruption of mitochondrial membrane potential ( m ) is one of the earliest intracellular events that occur following the onset

Fig. 8. LC–MS total ion chromatogram of the methanol root extract of Gentiana kurroo. Compounds: 1 (RT = 1.99) Loganic acid, 2 (RT = 6.79) Sweroside, 3 (RT = 7.39) Swertiamarin, 4 (RT = 8.06) Gentiopicroside, 5 (RT = 11.62) Gentisin, 6 (RT = 12.82) Isogentisin, 7 (RT = 14.14) Gentioside, 8 (RT = 15.01) Norswertianolin, 9 (RT = 15.87) Swertianolin, 10 (RT = 17.56) 4 -O-␤-d-glucosyl-6 -O-(4-O-␤-d-glucosylcaffeoyl)-linearoside, 11 (RT = 18.22) Swertisin.

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Fig. 9. Molecular structures of the various identified compounds in the methanol root extract of G. kurroo.

B.A. Wani et al. / Phytomedicine 20 (2013) 723–733

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Fig. 9. (Continued)

of apoptosis (Qi et al. 2010). Flow cytometric analysis of MiaPaCa2 cells after treatment with different concentrations of G. kurroo methanolic extract for 48 h, reveals that there is reduction in mitochondrial membrane potential ( m ). Compared to untreated control cells, incubation of MiaPaCa-2 cells with G. kurroo extract caused an obvious decrease of ( m ) after 48-h incubation in a concentration dependent manner (Fig. 7). G. kurroo extract induces loss of ( m ) from 75.6% in case of control cells to a low of 10.5% in case of 100 ␮g/ml extract. Loss of mitochondrial membrane potential is largely due to activation of mitochondrial permeability transition pore which leads to the subsequent release of Cytochrome C from mitochondria and consequently triggers other apoptotic factors (Kroemer et al. 1997). It has been earlier shown that the interaction of anticancer agents with mitochondria results

in an increase of the permeability of the inner mitochondrial membrane (Fulda et al. 1998). LC–ESI-MSMS analysis The phytochemical fingerprinting of the G. kurroo root extract was carried out by LC–ESI-MS. The extract was run under both positive and negative ESI-MS conditions and it showed several major and minor ionic species. The total ion MS chromatogram (TIC) is shown in Fig. 8. Fragmentation of the major peaks was used for the identification of compounds. The identification of the chemical compounds was also carried out by comparing the molecular ion peaks along with the MS fragmentation pattern with those of the literature (Urbain et al. 2009; Szucs et al. 2002).

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Table 2 Identified compounds in the methanolic root extract of Gentiana kurroo by LC–ESIMSMS. S. no

Compound

Measured mass

Retention time

Area sum%

1 2 3 4 5 6 7 8 9 10

Loganic acid Sweroside Swertiamarin Gentiopicroside Gentisin Isogentisin Gentioside Norswertianolin Swertianolin 4 -O-␤-d-glucosyl6 -O-(4-O-␤-dglucosylcaffeoyl)linearoside Swertisin

376.136 358.125 374.120 356.112 256.072 256.072 552.145 421.076 435.091 1008.310

1.99 6.79 7.39 8.06 11.62 12.82 14.14 15.01 15.87 17.56

11.09 4.45 1.43 24.23 0.36 20.21 17.14 11.05 0.53 5.52

446.120

18.22

5.52

11

The chemical constituents identified were Loganic acid, Sweroside, Swertiamarin, Gentiopicroside, Gentisin, Isogentisin, Gentioside, Norswertianolin, Swertianolin, 4 -O-␤-d-glucosyl-6 -O-(4-O-␤-dglucosylcaffeoyl)-linearoside and Swertisin (Fig. 9 and Table 2). The phytochemicals present in most Gentiana species have been found to be iridoids, xanthones, mangiferin and C-glucoflavones. The iridoids (mainly secoiridoid glucosides) appear to be present in all species investigated with a predominance of swertiamarin, sweroside and gentiopicroside. Xanthones are not universally present in Gentianaceae. The distribution of iridoids has been shown to have considerable value as a systematic character since they occur almost exclusively in Gentiana species. Plants from the Gentianaceae are best known for their bitter taste, which can be correlated with their content of iridoids. Bitter principles have been used in traditional remedies for the loss of appetite and fever, and are still included in many “tonic” formulations (Rodriguez et al. 1998).

Conclusion The extract shows potential antioxidant properties in different model systems, protects DNA from oxidative damages. The extract exhibited potential anticancer activity by inducing apoptosis in human pancreatic cancer cell line (MiaPaCa-2) through the disruption of mitochondrial membrane potential and disruption of mitochondrial membrane potential. LC–ESIMSMS fingerprinting of the extract showed that the extract consists of mainly of Loganic acid, Sweroside, Swertiamarin, Gentiopicroside, Gentisin, Isogentisin, Gentioside, Norswertianolin, Swertianolin, 4 -O-␤-d-glucosyl-6 -O-(4-O-␤-d-glucosylcaffeoyl)linearoside and Swertisin.

Conflict of interest The authors declare that there is no conflict of interest.

Acknowledgements Bilal A. Wani would like to thank University Grants Commission (UGC), India for providing research fellowship. Manzoor A. Rather would like to thank Council of Scientific and Industrial Research (CSIR), India for providing the Senior Research Fellowship (SRF). M.A.R would also like to acknowledge the help from Manoj Kushwaha for LC-MS/HPLC analysis. Akhtar H, Malik is acknowledged for the identification of the plant material.

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